Recent evidence indicates that a single G-protein-coupled receptor is responsible for the major biological effects of parathyroid hormone (PTH) and PTH-related protein (PTHrP). Acting through this receptor, these peptides play essential physiological roles- PTH as a major endocrine regulator of plasma calcium and skeletal homeostasis, and PTHrP as a growth and differentiation factor required for normal endochondral bone development. Moreover, when appropriately administered, PTH produces anabolic effects on the skeleton and may have therapeutic value in patients with osteopenic disorders. For these reasons, it is important to gain a detailed understanding of how the PTH/PTHrP receptor transmits instructive signals into target cells, and how signal transmission is regulated. Previous studies have demonstrated that signal transduction by the PTH/PTHrP receptor involves activation of two independent pathways- adenylyl cyclase/cAMP and phospholipase C/Ca-i2+. These pathways are subject to loss of responsiveness to PTH or PTHrP following exposure to agonists either acutely (resulting in receptor desensitization) or chronically (resulting in receptor down-regulation). Both processes are thought to be important determinants of target cell responsiveness, but almost nothing is known about their molecular bases. Recent studies demonstrate that the large cytoplasmic tail of the PTH/PTHrR receptor contains important determinants of receptor expression, endocytosis, and phosphorylation, suggesting an important role in the regulation of receptor function. Mechanisms of receptor regulation will be defined by: 1) determining the role of the cytoplasmic tail of the PTH/PTHrP receptor in receptor phosphorylation and homologous desensitization. Expression of recombinant, mutated PTH/PTH rP receptors in human embryonic kidney 293 cells will be used as a model system to map the sites in the tail that are required for PTH-stimulated phosphorylation and desensitization; 2) evaluating the role of G-protein-coupled receptor kinases (GRKs) in PTH/PTHrP receptor phosphorylation and desensitization. The role of a know GRK (beta-adrenergic receptor kinase, beta-ARK) will be tested by expression of a dominant inhibitory form of beta-ARK in PTH-responsive UMR 106 osteoblastic osteosarcoma cells, and potentially novel GRKs in these cells will be examined by reverse transcriptase-polymerase chain reaction (RT-PCR); 3) assessing the role of the C-terminal tail in PTH/PTHrP receptor endocytosis and recycling. Expression/mutagenesis will be used to identify specific sequences that regulate receptor trafficking, and to determine their role in agonist-stimulated receptor down-regulation; and 4) defining the molecular basis of the polarized expression of the PTH/PTHrP receptor by expressing and localizing the receptor in Madin- Darby canine kidney (MDCK) epithelial cells. Successful completion of these studies will provide mechanistic insights into how the biological effects of PTH and PTHrP are regulated at the level of their common receptor.